Kinetic properties of human dopamine sulfotransferase (SULT1A3) expressed in prokaryotic and eukaryotic systems: Comparison with the recombinant enzyme purified from Escherichia coli

Sulfation, catalyzed by members of the sulfotransferase enzyme family, is a major metabolic pathway which modulates the biological activity of numerou s endogenous and xenobiotic chemicals. A number of these enzymes have been expressed in prokaryotic and eukaryotic systems to produce protein for bioc hemical and physical characterization. However, the effective use of hetero logous expression systems to produce recombinant enzymes for such purposes depends upon the expressed protein faithfully representing the "native" pro tein. For human sulfotransferases, little attention has been paid to this d espite the widespread use of recombinant enzymes. Here we have validated a number of heterologous expression systems for producing the human dopamine- metabolizing sulfotransferase SULT1A3, including Escherichia coli, Saccharo myces cerevisiae, COS-7, and V79 cells, by comparison of K-m values of the recombinant enzyme in cell extracts with enzyme present in human platelets and with recombinant enzyme purified to homogeneity following E. coli expre ssion. This is the first report of heterologous expression of a cytosolic s ulfotransferase in yeast. Expression of SULT1A3 was achieved in all cell ty pes, and the K-m for dopamine under the conditions applied was approximatel y 1 mu M in all heterologous systems studied, which compared favorably with the value determined with human platelets. We also determined the subunit and native molecular weights of the purified recombinant enzyme by SDS-PAGE , electrospray ionization mass spectrometry, dynamic light scattering, and sedimentation analysis. The enzyme purified following expression in E. coli existed as a homodimer with M-r approximately 68,000 as determined by ligh t scattering and sedimentation analysis. Mass spectrometry revealed two spe cies with experimentally determined masses of 34,272 and 34,348 which corre spond to the native protein with either one or two 2-mercaptoethanol adduct s. We conclude that the enzyme expressed in prokaryotic and eukaryotic hete rologous systems, and also purified from E. coli, equates to that which is found in human tissue preparations, (C) 1999 Academic Press.